Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin

Abstract

Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi͞o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltagegated Ca 2؉ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.A major challenge in understanding the relationship between neural activity and development and between neuronal circuit activity and specific behaviors is to be able to control the activity of large populations of neurons or regions of individual nerve cells simultaneously. Recently, it was demonstrated that neuronal circuits can be manipulated by expressing mutated ion channels or G protein-coupled receptors (GPCRs). For example, the regional expression of a genetically modified K ϩ channel in Drosophila was able to reduce the excitability of targeted cells (i.e., muscle, neurons, photoreceptors) (1). Silencing of cortical neurons was achieved by binding of the peptide allostatin to its exogenously expressed receptor (2). Recently, Zemelman et al. (3) elegantly demonstrated that light activation of the protein complex, encoded by the Drosophila photoreceptor genes (i.e., arrestin-2, rhodopsin, and G protein ␣ subunit), could induce action potential firing of hippocampal neurons. Activation and deactivation of neuronal firing could also be achieved when ligand-gated ion channels, such as the capsaicin receptor, menthol receptor, purinergic receptors, or lightcontrollable K ϩ channel blockers, were used to control firing in hippocampal neurons (4, 5). However, the application of these techniques to control neuronal function especially in neural circuits and living animals is limited by their relatively slow time course, the complex...

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

“…S ince the initial description of light activation of excitable cells by Channelrhodopsin-2 (ChR2), both in cultured mammalian neurons [1][2][3] and in vivo in Caenorhabditis elegans (C. elegans) 4 , optogenetics has revolutionized research in various biological fields [5][6][7][8][9] . Optogenetics combines genetically encoded, light-sensitive proteins and photostimulation for exogenous control of specific cell functions 1,2,10 in organotypic cultures and intact animals 4,11,12 .…”

Selective Manipulation of Neural Circuits

“…Although this system made it easy to apply optogenetics to free-moving animals, it is still invasive-wireless headstagemounted systems still need to implant optical fiber into the brain. To reduce invasiveness channels that respond to longer wavelength light have been developed; longer wavelength light penetrates tissue more deeply than short [135][136][137][138][139][140]. Red light-activated opsins such as excitatory red lightactivated channelrhodopsin and inhibitory red-shifted cruxhalorhodopsin (Jaws) can be applied to the surface of the brain or through a thinned skull [119,120].…”

Next-Generation Optical Technologies for Illuminating Genetically Targeted Brain Circuits

“…Photons are absorbed by the all-trans-retinal cofactor of ChR2 that is endogenously present at sufficient levels in vertebrate CNS tissue (Fig. 3) (Li et al, 2005;Bi et al, 2006;Ishizuka et al, 2006;Zhang et al, 2006). Therefore, this technology is more suitable for use within intact living large animals such as mammals than uncaging-based methods that require the addition of large quantities of custom cofactors.…”